Intrathecal Delivery of Viruses in a Mouse Model for Treating Central Nervous System Diseases

Abstract

Source: Li, D., et al. Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice. J. Vis. Exp. (2019)

This video demonstrates the procedure for the intrathecal injection of adeno-associated viruses (AAV) into the cerebrospinal fluid (CSF) of an awake mouse, covering syringe preparation, spinal alignment, and precise needle insertion. This method validates targeted gene delivery, supporting its potential for CNS therapies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of 20% Lidocaine Hydrochloride Stock Solution

1. Weigh 2 g of lidocaine hydrochloride. Add 5–6 mL of sterile water and vortex gently. Increase the volume to 10 mL with sterile water.
2. 1.2 Filter the stock solution through 0.2-micron filters. Aliquot the stock solution, 1 mL per 1.5 mL microcentrifuge tube, and seal it with a sealing film. Store at 4 °C.

2. Direct Intrathecal AAV Delivery in Awake Mice

1. Wipe the work area using sterile gauze with 70% ethanol and prepare the required supplies.
2. Prepare 100 µL of AAVrh.10/1% lidocaine hydrochloride complex by adding 95 µL of rAAVrh10 stock solution (5 x 10¹² genome copies/mL) into a sterile 200 µL microcentrifuge tube, and add 5 µL of 20% lidocaine hydrochloride stock solution. Mix well by pipetting up and down. Then, store the virus solution on ice (4 °C).
   NOTE: 8 µL per mouse will be used.
3. Preparing the syringe with AAV solution for injection
   1. Assemble a 25 µL Hamilton syringe with a 27 G needle and align the beveled tip of the needle with the volumetric scale on the syringe.
   2. Draw 8 µL with 4 x 10¹⁰ genome copies of the virus solution into the syringe gently. Make sure to remove air bubbles.
4. Preparing the mouse
   NOTE: This study used male or female FVB/NJ mice (30–70 days old). IT injection was performed in the hood.
   1. Sterilize the work area in a hood with 70% ethanol. Put the awake mouse (male or female, 30–70 days old, 13–20 g weight) on a bedpiece in a prone position in the hood. Cover the upper body with sterile gauze to calm the mouse and avoid being bitten.
   2. Fix the animal by gripping appropriately and firmly on its pelvic girdle with a thumb on one side and forefinger/middle finger on the other side. Keep the skin between the bilateral pelvic girdles taut with the thumb and forefinger. Hold gently on the upper body of the animal with the palm.
   3. Shave the fur on its back between the bilateral pelvic girdles, then sterilize the skin surface with an iodide-based scrub and 70% ethanol.
5. Intrathecal Injection
   1. Feel the intervertebral space along the middle line between the bilateral pelvic girdles with a thumb or forefinger of the other hand and press an indentation with a fingernail to indicate the L5-L6 intervertebral space (locate the injection site).
   2. Rotate the base of the tail slightly and gently to indicate the midline of the spine. Adjust the needle bevel towards the animal's head before injection.
   3. Make sure that the animals are fixed firmly, and align the needle along the midline of the spine.
   4. Insert the needle gently and vertically (or tilt slightly 70–80°) in the intersection of the indentation, keeping the syringe in a central sagittal plane. Slowly reduce the angle to approximately 30° when it connects the bone, then slip the needle into the intervertebral space.
      NOTE: An evident sudden tail flick is a sign of successful entry into the intradural space. Once the needle enters the intervertebral space, the needle tip will feel firmly clamped. The 27 G needle used in this study is suited for IT delivery in mice but not rats.
   5. Inject the vector solution. Start the timer and inject 8 µL of the vector solution at a speed of 1 µL/4 s. Retain the needle approximately 1 minute after finishing delivery. Withdraw the needle with gentle rotation to avoid leaking.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FVB/NJ mice	Charles River Laboratories China
Lidocaine hydrochloride monohydrate	HEOWNS	73-78-9
AAV	Viral Vector Core of the Gene Therapy Center at University of Massachusetts Medical School
25µL Hamilton syringe/27-30g needle	GASTIGHT	1702
70% Ethanol	WEN ZHI
Gauze	Wei AN	5171112	8cm*10cm*12cm